Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Front Immunol ; 14: 1034032, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36845124

RESUMEN

Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.


Asunto(s)
Descubrimiento de Drogas , Proteínas Tirosina Quinasas Receptoras , Animales , Humanos , Ratones , Molécula de Adhesión Celular Epitelial , Distribución Tisular , Microscopía Fluorescente/métodos , Fosforilación
2.
Dev Cell ; 58(24): 2881-2895.e7, 2023 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-37967560

RESUMEN

Generating organs from stem cells through blastocyst complementation is a promising approach to meet the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here, we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation, we rescued heart and vascular system development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.


Asunto(s)
Sistema Cardiovascular , Corazón , Células Madre Pluripotentes , Animales , Ratones , Ratas , Blastocisto , Células Endoteliales , Miocitos Cardíacos , Corazón/embriología , Sistema Cardiovascular/embriología
3.
Int Rev Cell Mol Biol ; 372: 1-54, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36064262

RESUMEN

At the fundamental level, messenger RNA (mRNA)-based therapeutics involves the delivery of in vitro-transcribed (IVT) mRNA into the cytoplasm of a target cell, where it is translated into the desired protein. IVT mRNA presents various advantages compared to DNA and recombinant protein-based approaches that make it ideal for a broad range of therapeutic applications. IVT mRNA, which is translated in the cytoplasm after transfection into cells, can encode virtually any target protein. Notably, it does not enter the nucleus, which avoids its integration into the genome and the risk of insertional mutagenesis. The large-scale production of IVT mRNA is less complex than production of recombinant proteins, and Good Manufacturing Practice-compliant mRNA production is easily scalable, ideally poising mRNA for not only off-the-shelf, but more personalized treatment approaches. IVT mRNA's safety profile, pharmacokinetics, and pharmacodynamics, including its inherent immunostimulatory capacity, can be optimized for different therapeutic applications by harnessing a wide array of optimized sequence elements, chemical modifications, purification techniques, and delivery methods. The value of IVT mRNA was recently proved during the COVID-19 pandemic when mRNA-based vaccines outperformed the efficacy of established technologies, and millions of doses were rapidly deployed. In this review, we will discuss chemical modifications of IVT mRNA and highlight numerous preclinical and clinical applications including vaccines for cancer and infectious diseases, cancer immunotherapy, protein replacement, gene editing, and cell reprogramming.


Asunto(s)
COVID-19 , Neoplasias , COVID-19/terapia , Humanos , Inmunoterapia , Pandemias , Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo
4.
JCI Insight ; 7(22)2022 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-36227687

RESUMEN

Acute graft versus host disease (aGvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT) inflicted by alloreactive T cells primed in secondary lymphoid organs (SLOs) and subsequent damage to aGvHD target tissues. In recent years, Treg transfer and/or expansion has emerged as a promising therapy to modulate aGvHD. However, cellular niches essential for fostering Tregs to prevent aGvHD have not been explored. Here, we tested whether and to what extent MHC class II (MHCII) expressed on Ccl19+ fibroblastic reticular cells (FRCs) shape the donor CD4+ T cell response during aGvHD. Animals lacking MHCII expression on Ccl19-Cre-expressing FRCs (MHCIIΔCcl19) showed aberrant CD4+ T cell activation in the effector phase, resulting in exacerbated aGvHD that was associated with significantly reduced expansion of Foxp3+ Tregs and invariant NK T (iNKT) cells. Skewed Treg maintenance in MHCIIΔCcl19 mice resulted in loss of protection from aGvHD provided by adoptively transferred donor Tregs. In contrast, although FRCs upregulated costimulatory surface receptors, and although they degraded and processed exogenous antigens after myeloablative irradiation, FRCs were dispensable to activate alloreactive CD4+ T cells in 2 mouse models of aGvHD. In summary, these data reveal an immunoprotective, MHCII-mediated function of FRC niches in secondary lymphoid organs (SLOs) after allo-HCT and highlight a framework of cellular and molecular interactions that regulate CD4+ T cell alloimmunity.


Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Ratones , Animales , Linfocitos T Reguladores , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas/métodos
5.
Commun Biol ; 4(1): 799, 2021 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-34172833

RESUMEN

The presence of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with an unfavorable prognosis. FLT3 inhibitors, such as midostaurin, are used clinically but fail to entirely eradicate FLT3-ITD + AML. This study introduces a new perspective and highlights the impact of RAC1-dependent actin cytoskeleton remodeling on resistance to midostaurin in AML. RAC1 hyperactivation leads resistance via hyperphosphorylation of the positive regulator of actin polymerization N-WASP and antiapoptotic BCL-2. RAC1/N-WASP, through ARP2/3 complex activation, increases the number of actin filaments, cell stiffness and adhesion forces to mesenchymal stromal cells (MSCs) being identified as a biomarker of resistance. Midostaurin resistance can be overcome by a combination of midostaruin, the BCL-2 inhibitor venetoclax and the RAC1 inhibitor Eht1864 in midostaurin-resistant AML cell lines and primary samples, providing the first evidence of a potential new treatment approach to eradicate FLT3-ITD + AML.


Asunto(s)
Citoesqueleto de Actina/fisiología , Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Estaurosporina/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Citoesqueleto de Actina/química , Apoptosis/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pironas/farmacología , Quinolinas/farmacología , Estaurosporina/farmacología , Sulfonamidas/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/fisiología
6.
Front Immunol ; 11: 593321, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33584657

RESUMEN

Acute graft-versus-host disease (aGvHD) is a severe and often life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). AGvHD is mediated by alloreactive donor T-cells targeting predominantly the gastrointestinal tract, liver, and skin. Recent work in mice and patients undergoing allo-HCT showed that alloreactive T-cells can be identified by the expression of α4ß7 integrin on T-cells even before manifestation of an aGvHD. Here, we investigated whether the detection of a combination of the expression of T-cell surface markers on peripheral blood (PB) CD8+ T-cells would improve the ability to predict aGvHD. To this end, we employed two independent preclinical models of minor histocompatibility antigen mismatched allo-HCT following myeloablative conditioning. Expression profiles of integrins, selectins, chemokine receptors, and activation markers of PB donor T-cells were measured with multiparameter flow cytometry at multiple time points before the onset of clinical aGvHD symptoms. In both allo-HCT models, we demonstrated a significant upregulation of α4ß7 integrin, CD162E, CD162P, and conversely, a downregulation of CD62L on donor T-cells, which could be correlated with the development of aGvHD. Other surface markers, such as CD25, CD69, and CC-chemokine receptors were not found to be predictive markers. Based on these preclinical data from mouse models, we propose a surface marker panel on peripheral blood T-cells after allo-HCT combining α4ß7 integrin with CD62L, CD162E, and CD162P (cutaneous lymphocyte antigens, CLA, in humans) to identify patients at risk for developing aGvHD early after allo-HCT.


Asunto(s)
Biomarcadores , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Enfermedad Aguda , Animales , Antígenos CD/metabolismo , Biopsia , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Enfermedad Injerto contra Huésped/diagnóstico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Ratones , Pronóstico , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Trasplante Homólogo
7.
Stem Cell Res ; 36: 101410, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30878013

RESUMEN

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40.


Asunto(s)
Antígeno AC133/genética , Línea Celular , Células Madre Pluripotentes Inducidas , Animales , Diferenciación Celular , Técnicas de Reprogramación Celular , Células Clonales , Sangre Fetal/citología , Marcadores Genéticos , Humanos , Cariotipo , Ratones Endogámicos NOD , Ratones SCID , Virus Sendai
8.
Stem Cell Res ; 33: 125-129, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30343102

RESUMEN

Islet-1 (Isl1) is a transcription factor essential for life expressed in specific cells with different developmental origins. We have generated iPSC lines from fibroblasts of the transgenic Ai6 x Isl1-Cre (Ai6IslCre) mouse. Here we describe the complete characterization of four iPSC lines: ATCi-Ai6IslCre10, ATCi-Ai6IslCre35, ATCi-Ai6IslCre74 and ATCi-Ai6IslCre80.


Asunto(s)
Proteínas con Homeodominio LIM/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones
9.
Stem Cell Res ; 21: 1-4, 2017 05.
Artículo en Inglés | MEDLINE | ID: mdl-28677526

RESUMEN

We generated ATCi-MF1 induced pluripotent stem (iPS) cell line from Macaca fascicularis adult skin fibroblasts using non-integrative Sendai viruses carrying OCT3/4, KLF4, SOX2 and c-MYC. Once established, ATCi-MF1 cells present a normal karyotype, are Sendai virus-free and express pluripotency associated markers. Microsatellite markers analysis confirmed the origin of the iPS cells from the parental fibroblasts. Pluripotency was tested with the in vivo teratoma formation assay. ATCi-MF1 cell line may be a useful primate iPS cell model to test different experimental conditions where the use of human cells can imply ethical issues, as microinjection of pluripotent stem cells in pre-implantational embryos.


Asunto(s)
Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Virus Sendai , Piel/metabolismo , Factores de Transcripción , Transducción Genética , Animales , Línea Celular , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Macaca fascicularis , Piel/citología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética
10.
Stem Cell Res ; 16(3): 617-21, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27346195

RESUMEN

Mef2c Anterior Heart Field (AHF) enhancer is activated during embryonic heart development and it is expressed in multipotent cardiovascular progenitors (CVP) giving rise to endothelial and myocardial components of the outflow tract, right ventricle and ventricular septum. Here we have generated iPSC from transgenic Mef2c-AHF-Cre x Ai6(RCLZsGreen) mice. These iPSC will provide a novel tool to investigate the AHF-CVP and their cell progeny.


Asunto(s)
Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Miocardio/citología , Animales , Diferenciación Celular , Reprogramación Celular , Cuerpos Embrioides/citología , Cuerpos Embrioides/metabolismo , Genotipo , Células Madre Pluripotentes Inducidas/metabolismo , Cariotipo , Factores de Transcripción MEF2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Teratoma/metabolismo , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA